The sorting signal motif for targeting pro-opiomelanocortin (POMC, pro-ACTH/endorphin) and pro- enkephalin (pro-Enk) to the regulated secretory pathway was identified. For POMC, the motif consists of a 13 amino acid amphipathic loop stabilized by one disulfide bridge located at the N-terminus of POMC. Two hydrophobic and two acidic residues were on the surface of this loop and mutagenesis studies showed that they were important for the sorting function of the signal. A similar 19 amino acid amphipathic loop sorting signal was also identified at the N-terminus of pro-Enk, indicating that these motifs represent consensus sorting signals. A sorting receptor that is specific for the sorting signal of POMC has been identified in bovine pituitary Golgi and secretory granule membranes. Binding studies showed that other regulated pathway proproteins e.g. pro-Enk, pro-insulin and chromogranin A also bound to the receptor and were displaced by N-POMC1-26 which contains the POMC sorting signal motif. However, constitutively secreted proteins did not bind. The receptor has been purified, cloned and antisense studies indicate that it functions in vivo to sort prohormones to the regulated secretory granules for processing and secretion. The novel class of prohormone converting aspartic proteases was studied. One member of this class, yeast aspartic protease 3 (YAP3p) has been shown to cleave at mono- and paired basic residue sites of mammalian prohormones. Specificity studies on YAP3p showed an enhancement in efficiency of cleavage of prohormones having additional basic residues upstream and/or downstream of the cleavage site. Substrates with an Arg basic residue in the P2' position was the most efficiently cleaved. Modeling studies revealed an acidic residue, Asp in the S2' pocket which may explain this increased efficiency. Biosynthesis studies showed that YAP3p is synthesized as a pro-enzyme which is autocatalytically cleaved to form the active enzyme. It can be further cleaved at an Asn residue to yield alpha and beta subunits which are then linked by a disulfide bridge to form a heterodimer. Antibodies against YAP3p were shown to cross-react with the purified, bovine intermediate pituitary POMC converting enzyme (PCE) an 70 kD basic residue-specific aspartic protease, indicating homology between them. In addition, a about 90 kDa anti- YAP3p immunoreactive protein was found in hypothalamus and anterior pituitary secretory granules. The about 90 kD YAP3-related protein was distributed in peptide-rich brain regions and co-localized with CCK, and vasopressin mRNA in rat brain neurons. Thus, YAP3p-related aspartic proteases, including PCE, likely play an important role in prohormone processing in mammalian brain. Several clones have now been obtained using the YAP3p antibody to screen a mouse brain expression library and they are currently being sequenced.